The overall objective of this project is to understand the cellular and molecular mechanisms responsible for the specification, patterning, and differentiation of internal organs during development. More specifically, how the elaborate network of blood vessels arises during vertebrate embryogenesis. Blood vessels are ubiquitous and vital components of all vertebrate animals, innervating and supplying every tissue and organ with oxygen, nutrients, and cellular and humoral factors. They have also become a subject of great clinical interest in recent years, particularly with the potential shown by antiangiogenic therapies for combating cancer. Many of our insights into mechanisms of blood vessel formation have come from developmental studies. The zebrafish, a small tropical freshwater fish, possesses a unique combination of features that make it particularly well suited for studying blood vessels. The fish is a genetically tractable vertebrate with a physically accessible, optically clear embryo. These features provide a tremendous advantage for studying vascular development- they permit observation of every vessel in a living animal and simple, rapid screening for even subtle vascular-specific mutants. Major aims of the laboratory include: (i) Developing experimental tools and resources to enhance the zebrafish as an experimental model for studying vascular embryogenesis, (ii) Studying the molecular basis for arterial-venous differentiation, (iii) Studying the basis for vascular patterning and morphogenesis (iv) Forward-genetic analysis to identify novel zebrafish genes critical for vascular development Developing Tools for Experimental Analysis of Vascular Development in the Zebrafish An important aim of this project has been to develop new experimental tools for studying blood vessel formation in this organism. To this end, we previously established a microangiographic method for imaging patent blood vessels in the zebrafish and used this method to compile a comprehensive staged atlas of the vascular anatomy of the developing fish (http://eclipse.nichd.nih.gov/nichd/lmg/redirect.html). We also generated transgenic zebrafish lines expressing green fluorescent protein (GFP) in vascular endothelial cells (VEC), making it possible for us to visualize the blood vessel formation in intact, living embryos. We have developed methodologies for long-term multiphoton confocal timelapse imaging of the dynamics of blood vessel formation in these transgenic fish, and we have used these methods to examine the morphogenesis of developing trunk and cranial vessels and adult fin blood vessels.. An important current priority in the laboratory is the development of additional transgenic lines that permit dynamic visualization of specific subsets of vessels such as arteries and veins, or subcellular structures within vascular cells (see below), or that permit temporally regulated gene expression within the vasculature. Molecular Dissection of Arterial-Venous Development We have uncovered a molecular pathway regulating the acquisition of arterial-venous identity. Although the fundamental distinction between these two types of blood vessels has been appreciated for thousands of years, the fact that arterial and venous endothelial cells have distinct molecular and functional identities has only become apparent very recently, and the mechanisms responsible for establishing this identity have not been elucidated. We have shown that sonic hedgehog (SHH), vascular endothelial growth factor (VEGF), and notch signaling act in series to determine arterial venous identity. We were able to do this by using different combinations of drug treatments, mutants, morpholinos, and mRNA injections to activate or repress the activity of each of these signals in vivo, either alone or in combination. Subsequent studies from other labs have supported a similar role for this novel pathway during mammalian arterial differentiation. Using genetic screening and positional/candidate cloning methods we recently identified ?y10?, a mutant in phospholipase C gamma-1, and used this to demonstrate that this gene is a major downstream effector of VEGF signaling in vivo. We are continuing to use genetic screening and other methods such as microarray analysis to identify and characterize additional genes in the arterial differentiation pathway. Analysis of Vascular Patterning and Morphogenesis An important research area has been studying the mechanisms and molecular basis for vascular patterning and morphogenesis during development, in particular how vascular networks assemble with a defined, stereotypic anatomical pattern. We have used multiphoton time-lapse imaging of the developing trunk to obtain important new insights into how angiogenic networks assemble in vivo. These insights include elucidation of a novel 2-step model for vascular network formation and evidence for genetic determination of vascular pattern. Additional studies have allowed us to define some of these genetic pattern determination factors, showing that well-known neuronal guidance factors also play an important role in vascular guidance and vascular patterning. We have recently found that Semaphorin-Plexin signaling it is an essential determinant of trunk vessel pattern, uncovering a Plexin gene expressed specifically in the vasculature. In the developing trunk, angiogenic intersegmental vessels extend near somite boundaries. Loss of Plexin function via morpholino injection or genetic mutation causes dramatic mispatterning of these vessels, which are no longer restricted to growth near intersomitic boundaries. Somites flanking intersegmental vessels express Semaphorins and reducing the function of these Semaphorins also causes intersegmental vessel mispatterning. These results indicate that the establishment of anatomical pattern in the developing vasculature is directed in part by cues and mechanisms similar to those used to pattern the developing nervous system, including Semaphorin-Plexin signaling. We are currently attempting to extend these studies and uncover the signal transduction machinery that transmits Semaphorin-Plexin signaling. Using genetic screening methods, we are also identifying additional new loci that influence vascular patterning in vivo. Isolation and Analysis of Vascular-Specific Mutants Genetic dissection of vascular development and the molecular pathways that regulate it is an important ongoing goal of the UVO. We employ unbiased, forward genetic mutational screening approaches to identify and characterize zebrafish mutants that affect the formation of the developing vasculature. We have already positionally cloned the defective genes from a number of vascular patterning mutants, including violet beauregarde (defective in Alk1/acvrl1), y10 (defective in phospholipase C-gamma 1), and kurzschluss (defective in a novel chaperonin). We have performed new screens for vascular specific mutants using transgenic zebrafish expressing green fluorescent protein (GPF) in blood vessels. To date, these ongoing mutant screens have resulted in the identification of over 30 new vascular-specific mutants. The phenotypes of these mutants include too few vessels, too many vessels, increased sprouting/branching, and mispatterned vessels. Our experience suggests that these ongoing mutant screens should continue to yield a rich harvest of novel vascular-specific mutants and bring to light new pathways regulating the specification, differentiation, and patterning of the developing vertebrate vasculature.